Abstract Background: In order to assess the possibility that antibiotic resistance genes are being transferred from animals to environmental bacteria, non-enteric Ampicillin resistant (AmpR) bacteria were isolated from a cattle farm, a meat packing plant sewage lagoon, and the Mississippi river. Methods: Organisms were isolated on APT media containing 50 mg/L Amp, screened for cefinase activity, and the inability to ferment lactose to acid and gas in broth. MIC for Amp was determined using Etest  strips, and a profile of resistance to 17 antibiotics was determined using the Kirby-Bauer agar diffusion test. Chromosomal DNA was extracted by phenol:chloroform separation in the presence of CTAB detergent and by DNeasy . Plasmid extractions were performed with the Qiagen mini-prep kit  and the Wizard mini-prep kit . These DNAs were used in Southern hybridization experiments with probes for class A (TEM1-type) and class B (metallo-)  -lactamases. Six of the isolates were identified by sequencing of PCR amplified 16S rDNA (GenBank accession numbers). Results: A total of 17 non-enteric strains were studied, and 14 had MIC values greater than 256 mg/L. Pseudomonas sp. FDM13 (AY464123), from the sewage lagoon, contained plasmid DNA, but was not capable of transforming E. coli strains INV  F’ or XL10 Gold . No plasmid DNA was detected in the 16 isolates from the cattle farm and the Mississippi river. None of the chromosomal DNAs, or FDM13 plasmid DNA hybridized with the TEM1 probe. Pseudomonas sp. CPE30 (AY484469), Aeromonas sp. WC56 (AY484470), Morganella sp. CPD30 (AY464464), Pseudomonas sp. ACP14 (AY464463), and Chryseobacterium ACP12 (AY464462) showed the strongest hybridization with the metallo-β-lactamase probe. Conclusion: The lack of R-plasmids and the failure of hybridization with the TEM1 probe suggest that lateral gene transmission from enteric bacteria associated with animals to environmental bacteria is not taking place. On the other hand, environmental bacteria that show a high degree of resistance to Amp were widespread, and resistance in these bacteria may be due to zinc- hydrolases, or other yet unidentified resistance mechanisms.

Antibiotic Resistance Transfer in Agriculture Cow Manure Enteric Bacteria Fecal Coliform Use of antibiotics for feed and treatment selects for Ab R phenotype Class A TEM bla genes Non-enteric soil bacteria Lateral gene transfer – Conjugation, transformation, transduction Non-enteric soil bacteria Resistance evolved over time Class B metallo-  -lactamase genes ? ?

Investigation into Antibiotic Resistance in Bacteria in Agricultural Settings  Hypothesis 1 - Resistance due to lateral gene transfer – Resistance highly specific to antibiotics used – Resistance genes may be carried on plasmids –  -Lactamase gene may resemble class A TEM bla found in enterics  Hypothesis 2 (null) - Resistance evolved in soil microorganisms – Broader resistance to variety of antibiotics encountered in soil over time – Resistance may be plasmid or chromosomally encoded – Class B Metallo-  - Lactamase observed in Caulobacter may be present

Water Collection Sites  Meat Cattle farm in Swinton, MO. Antibiotic use reported as penicillin only.  100 ml water samples were taken from 3 ponds and a creek adjacent to the farm ( )  UTM 16 coordinates shown for map datum NAD 27 in CONUS.  ArcMap used to plot the points onto the topographic map.

Isolation Approach  Ampicillin resistance – Plated on APT agar w/ 50  g/ml ampicillin – Single colony taken from each plate with growth, unless additional morphotypes present – Screened for cefinase activity  Non-coliform status (accepted if one of the following are true) – Gram positive – No lactose fermentation on EMB – No gas from lactose broth

Organisms IsolateSourceTentative IdentificationGenBank Accession Number ACP12Cattle Pond 1ChryseobacteriumAY ACP14Cattle Pond 1PseudomonasAY CPA20Cattle Pond 2 CPA30Cattle Pond 3PseudomonasNot yet prepared CPB30Cattle Pond 3 CPC20Cattle Pond 2PseudomonasNot yet prepared CPC32Cattle Pond 3 CPC30Cattle Pond 3 CPD30Cattle Pond 3MorganellaAY CPD32Cattle Pond 3Escherichia senegalensisNot yet prepared CPE30Cattle Pond 3PseudomonasAY WC20Wolf Creek WC24Wolf Creek WC42Wolf Creek WC56Wolf CreekAeromonasAY MR55Mississippi River FDM13Meat-packing plant sewagePseudomonas syringiaeAY464123

Documentation of Resistance  MIC of Ampicillin for isolates was determined with Etest  strips (upper left) – Result: 14 of the isolates had a MIC of greater than 256 µg/ml. CPB30 (96µg/ml) and CPC32 (128 µg/ml) were slightly lower.  Kirby-Bauer Agar diffusion tests (upper right) were used to test for resistance to  - lactam (Oxacillin, Cefaclor, Cefazolin, Cefotaxime, Imipenem, Carbenicillin )and non-  -lactam antibiotics (Levaquin, tetracycline, Polymyxin B, Erythromycin, Kanamycin, Streptomycin, Rifampin, Novobiocin). – Result 1: All of the isolates were resistant to at least 1 non-  -lactam antibiotic, and 14 were resistant to 2 or more. – Result 2: None of the isolates showed resistance to imipenem, suggesting no metallo-  -lactamase activity.

Comparison of Frequency of Resistance to Various Antibiotics  Organisms used – Cattle farm isolates (organisms under study) – Reference organisms associated with soil (Lab teaching strains): B. cereus, B. megaterium, B. subtilis, B. brevis, B. pumilis, P. aeruginosa, P. putida, P. fluorescens, P. paucimobilis, P. stutzeri – Chat Pile Lead-mine tailings isolates (non-selected environmental isolates): 10 organisms including Rhodococcus, Pseudomonas, Streptomyces, Ochrobactrum, and Arthrobacter  Antibiotics used –  -lactam: Ampicillin, Carbenecillin, Cefazolin, Cephatoxime, Cefaclor – Non-  -lactam: Erythromycin, Kanamycin, Polymyxin B, Streptomycin, Tetracycline

Frequency of Resistance among Isolates by Antibiotic Group Organisms/AbSusceptibleIntermediateResistantTotal Reference/ β- lactam Reference/ Non-β-lactam Cattle Farm/β- lactam Cattle Farm/ Non-β-lactam Chat Pile/ β- lactam Chat Pile/Non- β-lactam Total

Chi-square Contingency Table Organism/AbSusceptibleIntermediateResistantTotal Reference / β-lactam a Reference / Non-β- lactam b Cattle Farm/β-lactam Cattle Farm/ Non-β- lactam Chat Pile/ β-lactam Chat Pile/Non-β-lactam Total c a significantly higher than expected, α=0.005, df=2, Fcrit= 10.6 b significantly lower than expected, α=0.005, df=2, Fcrit= 10.6 C significant variation among groups; α=0.005, df=10, Fcrit= 25.2

Antibiotic Testing Summary  Non-imepenem resistance implies no metallo-  - lactamase activity  Cattle farm isolates resistant to 3 or more classes of antibiotics suggesting exposure to more than just penicillin  Cattle farm isolates are more resistant to  -lactam than non-  -lactam antibiotics, suggesting a specific mechanism of resistance

Molecular Approaches  Isolate plasmids  Isolate chromosomal DNA  Southern Blot performed on each isolate using probes for TEM and metallo-  -lactamases

Plasmid DNA Isolation Studies  DNA was isolated from each bacterium, as well as FDM13 (an antibiotic resistant bacterium known to harbor plasmids). – Techniques used: Wizard miniprep (shown here), Qiagen spin kit, Qiagen miniprep kit – Smears likely due to glycosylated DNA, but no distinct bands  No plasmids detected λHind III FDM13 CPA20 ACP14 MR55 CPD32 WC24 CPC32 CPE30 ACP12 λHindIII WC24 CPD30 CPC30 CPC20 WC20 WC56 CPA30 CPB30 23kbp 4kbp 2kbp 500bp 23kbp 4kbp 2kbp 500bp

16S rDNA Hybridization - Control  Chromosomal DNA was obtained with DNeasy kit   16s rDNA hybridization was used to determine if DNA was suitable for hybridization.  RFLP can also be used to determine if some of the bacteria are similar or the same species. λ Hind III E.coli WC42 WC56 WC20 ACP14 ACP12 CPB30 CPC20 CPA30 CPC32 MR55 WC24 CPA20 CPE30 CPD32 CPC30 CPD30 λ Hind III 23kbp 2kbp 500bp 23kbp 2kbp 500bp

Metallo-ß-Lactamase Hybridization  EcoRI-digested Chromosomal DNA probed with a 1064 bp BstXI fragment of a putative metallo-β- lactamase from G. metallireducens (positive control)  ACP12 - 9kbp, WC56 - 3kbp, ACP14 - 9kbp and 8kbp, CPD30 - 8kbp and 6kbp, and CPE30 - 8kbp and 6kbp  E. coli (negative control), WC24, CPA30, MR55, CPA20, and CPD32 showed non specific hybridization.  CPB30, WC42, CPC20, CPC32, WC20, and CPC30, and reference strains showed no hybridization to this probe. λHindIII G. metallireducens ACP12 WC56 ACP14 CPD30 CPE30 E.coli 23kbp 4kbp 2kbp 500bp * * * * *

Lack of TEM1 Hybridization λHindIII Hem2b MR55 WC20 ACP14 CPA20 CPD30 CPC30 CPD32 CPE30 23kbp 4kbp 2kbp 500bp E. coli EcoRI Chromosomal DNA was probed with a 540 DdeI internal fragment of the bla gene from pBR322. Hem2B is plasmid DNA containing the bla gene (positive control). E. coli is negative control. No hybridization with this probe was seen with any ampicillin resistant laboratory strains.

Conclusion  Chromosomal DNA did not hybridize with TEM1 probe.  Interspecies gene transfer from enteric to environmental bacteria may not be occurring.  Bacteria in the environment are already resistant to antibiotics and are more competitive than the transient fecal organisms.

Conclusion  Resistance may be due to metallo-β- lactamase or some other unidentified mechanism.  Pseudomonas resistant to cefotaxime, this resistance may be due to chromosomal AmpC.

Acknowledgements  Principal funding for this project came from the Southeast Missouri State University Grants and Research Funding Committee.  Additional funding to support 16S rDNA sequencing costs came from the Southeast Missouri State University Undergraduate Research Program. Julie Rengel would like to thank Dr. Allan Bornstein and Dr. Jane Stephens for their support of undergraduate research.  Funding for student travel was made available through the Southeast Missouri State University Student Professional Development program (Drs. Rick Burns and Christina Frazier).  Co-authors not in attendance: Julie Rengel, Melanie Miller, Jennifer Arnold, and Josh Wolozynek  Kimberleigh Foster’s thesis Committee: Dr. Bjorn Olesen and Dr. Allen Gathman  Dr. Walt Lilly, Dr. John Scheibe, and Maija Bluma  Dave Bridges for help with ARC Map